The bacterium Escherichia coli has a single, circular chromosome that is replicated and segregated with great precision to daughter cells during cell division. Replication proceeds bi-directionally from a single origin and terminates on the opposite side of the chromosome. The relative simplicity of this system and the limited number of cell components required for its propagation make it a model system for DNA replication and segregation in general. We have developed a P1 parS GFP-ParB system for localization by fluorescent microscopy of any desired locus on the E. coli chromosome in living cells. Using similar DNA recognition systems of different specificities, we can now label up to three chromosomal loci simultaneously, using three differently colored fluorescent proteins. The technique works well in living cells and allows us to follow the fate of chromosomal sequences through several generations by time-lapse microscopy. In addition, we have used the technique, in combination with flow cytometry, to determine the spatial distributions of given loci at defined points in the cell cycle in a cell population. This effort has been greatly augmented by collaboration with the laboratory of Flemming Hansen, the Technical university of Denmark. With him, have developed automated methods for the measurement of the positions of fluorescent foci in the cells that permits accurate measurement of thousands of cells from microscopic images. We are also developing rapid methods for the analysis of the large data sets that we are able to collect. These methods provide us with powerful tools for the investigation of the replication and segregation dynamics of the chromosome. So far, we have been able to disprove the currently popular model for chromosome segregation involving simultaneous segregation of the bulk of the DNA. Rather, we show clearly that DNA is segregated progressively as it is replicated. Our investigations are revealing unexpected features of DNA organization and motion, including the fact that the two arms of the circular chromosome lie in opposite halves of the resting cell. We have been able to conclude that DNA segregation proceeds in concert with replication in a process that may resemble the formation of separable sister chromatids in higher organisms. In the past year, we have made substantial progress toward understanding chromosome segregation at fast growth rates, where the initiation of chromosome replication becomes uncoupled from the cell division cycle and the cells become functional diploids. Under these conditions, cell division occurs while chromosome replication is ongoing. We have confirmed that segregation is driven directly by replication so that segregation of chromosome domains can occur in generations previous to the one in which the regions are placed in separate cells by cell division. We are currently investigating the temporal transitions that occur as the newly replicated DNA emerges from the replication forks and and is organized into new nucleoid structures. We have recently obtained evidence that the SeqA protein binds specifically to newly replicated DNA to from an intermediate structure in this process. This structure appears to have the sister duplexes paired, thus delaying segregation of the chromosomal markers. The subsequent re-organization of the DNA both achieves segregation and forms the mature nucleoid structure. New advances that we have made in our image analysis software allow us to investigate the three-dimensional position of markers in large populations. Our initial analysis strongly suggests that the replicating nucleoid is a shell-like structure, with the newly replicated DNA on the inside and the unreplicated DNA on the outside. This is unexpected and is contrary to recent speculation. We believe that this structure indicates that an active mechanism for the placement of the newly replicated origins places them on the radial cell axis. The visible properties of DNA replication and segregation need to be linked to the biochemical and structural properties of the macromolecules involved in the key events. To date, we have made significant progress in understanding the role of the SeqA protein that is involved in both replication and segregation of the chromosome. In collaboration with Dr. Alba Guarne (McMaster University) we have recently solved the crystal structure of the entire SeqA protein in a complex with its cognate DNA sequence. Using the structure as a guide, we have constructed mutant proteins and have determined their effects on DNA replication and segregation. These studies have lead us to a working model for the roles of SeqA that is currently being tested. This year, we have brought to near completion the visualization of the SeqA protein in living cells and the study of the dynamics of its localization as the replication forks progress around the chromosome. This project has brought with it new challenges in data gathering and computational analysis that have been successfully addressed in our software development project.